Bio-functionalized nanocolloids of ZnS quantum dot/amine-rich polypeptides for bioimaging cancer cells with antibacterial activity: “seeing is believing”

Among almost 200 types of cancers, glioma is considered one of the most common forms of malignant tumors located in the central nervous system (CNS). Glioblastoma (GBM), one of the deadliest types of brain cancer, remains one of the challenges faced by oncologists. Thus, smartly designed nanomaterials biofunctionalized with polypeptides can offer disruptive strategies relying on the earliest possible diagnosis (“seeing is believing”) combined with more efficient therapies for fighting cancer cells. To worsen this scenario, bacteria infections very often pose a serious challenge to cancer-immunodeficient patients under chemotherapy. Thus, in this research, we report for the first time the design and synthesis of novel nanoconjugates composed of photoluminescent ZnS quantum dots (ZnS QDs), which were directly surface biofunctionalized with epsilon-poly-l-lysine (εPL), acting as an amine-rich cell-penetrating peptide (CPP) and antimicrobial peptide agent (AMP). These nanoconjugates (named ZnS@CPP–AMP) were produced through a one-step facile, eco-friendly, and biocompatible colloidal aqueous process to be applied as a proof of concept as nanoprobes for bioimaging GBM cancer cells (U87-MG) associated with synergic antibacterial activity. They were characterized regarding their physicochemical and optical properties associated with the biological activity. The results demonstrated that chemically stable aqueous colloidal nanoconjugates were effectively formed, resembling core–shell (inorganic, ZnS, organic, εPL) nanostructures with positively surface-charged features due to the cationic nature of the amine-rich polypeptide. More importantly, they demonstrated photoluminescent activity, cytocompatibility in vitro, and no significant intracellular reactive oxygen species (ROS) generation. These ZnS@CPP–AMP nanocolloids behaved as fluorescent nanoprobes for bioimaging GBM cancer cells, where the polycationic nature of the εPL biomolecule may have enhanced the cellular uptake. Additionally, they displayed mild antibacterial growth inhibition due to electrostatic interactions with bacterial membranes. Thus, it can be envisioned that these novel photoluminescent colloidal nanoconjugates offer novel nanoplatforms that can be specifically targeted with biomolecules for bioimaging to diagnose highly lethal cancers, such as GBM, and as an adjuvant in antibacterial therapy.

Next, the cells were scraped and incubated at 95 ˚C (Termomix, Eppendorf F1.5).After 60 min, the reaction was stopped in the ice bath for 5 min.Then, a volume of 300 µL was transferred to a 96-well plate, and the final pink-colored solution formed from the reaction between thiobarbituric acid and malondialdehyde (MDA) was subjected to absorbance analysis (Varioskan™ LUX multimode microplate reader, Thermo Scientific) at λ = 532 nm.The results were calculated as nmol of MDA-TBA/mg of cellular protein using 156 mM -1 cm -1 as a molar extinction coefficient of MDA-TBA.Proteins extracted from cells were calculated using the Bradford method (Bradford reagent, Sigma-Aldrich, USA) using bovine serum albumin (BSA, Sigma-Aldrich, USA) as reference material.Data were presented as the mean and standard deviation of three replicates (n = 3).
Next, 40 µL SDS solution/4 % HCl (37 %, Sigma-Aldrich, USA) was placed in each well and incubated at 37 ˚C/16 h/5% CO 2 .Then, the volume of 100 µL from each well was aspirated and transferred to a blank 96-well plate, and the absorbance was evaluated using iMark™ Microplate Absorbance Reader (Bio-Rad ® , USA) at λ = 595 nm.The percentage of cell viability was estimated according to Eq. S1 after blank corrections, and the values of the control group were set to 100% of cell viability.Data were presented as the mean and SD of six replicates (n = 6).
Digital images were captured in the sequence with a Ti-U epifluorescence microscope (Nikon Instruments, USA) using a FITC filter cube for ZnS green emission.To reveal late endosome/lysosome vesicles and cell nucleus, U87-MG cells after 30 min of incubation with ZnS@CPP-AMP nanoconjugate were washed and additionally stained with LysoTracker ® Deep Red (Thermo Fisher Scientific, USA) and DAPI (4′,6-diamidino-2-phenylindole, Thermo Fisher Scientific, USA), respectively, according to the manufacturer's protocol.For blue fluorescence emission, a DAPI filter cube was used.Images were acquired using a Texas Red filter cube for deep red-emission detection.Three-color confocal fluorescence images were recorded separately in the correspondent channel and merged afterward.

. Antibacterial Activity
To determine the antibacterial activity of PL solution and ZnS@CPP-AMP nanoconjugates, the minimum inhibitory concentration (MIC) was used using the agar microdilution method.
For the MIC protocol, from an initial concentration of the treatment (PL = 300 g/mL for both PL solution and ZnS@CPP-AMP), several concentrations were tested using a 2-fold dilution to determine the lowest concentration that inhibits the growth of a strain of bacteria.Samples were tested against the reference strains Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 29213).The antibiotic standards (ciprofloxacin, gentamycin, and tetracycline, Sigma-Aldrich, USA) were also tested against the reference strains to ensure that the results were within acceptable quality control limits for susceptibility testing according to Clinical and Laboratory Standards Institute (CLSI) guidelines [1].